Protein modification by ubiquitin (Ub) is a critical regulatory process for virtually all aspects of cell biology. Substrate proteins can be modified with single ubiquitin on one (monoubiquitylation) or multiple sites (multi-ubiquitylation). Alternatively, several rounds of ubiquitination can occur on ubiquitin itself, leading to the formation of a polyubiquitin chain. Any of the seven lysines, or the amino terminus, of ubiquitin can be used to polymerize ubiquitin (Peng et al., 2003), so there are a huge number of differently linked polyubiquitin signals that can be formed. Ub signals are reversible as ubiquitin can be removed from substrates by deubiquitinating enzymes. The diverse Ub signals are recognized in cells by a myriad of receptors that carry distinct ubiquitin binding motifs recognizing mono- or polyubiquitinated substrates (Hicke et al., 2005). Among the seven UbUb linkages found in cells, the best understood examples are Lys48- (K48-) and Lys63- (K63-) linked ubiquitin chains, which are considered as canonical linkage. Lys48-linked Ub chains usually targets modified substrates to the 26S proteasome for degradation (Pickart, 2004), and Lys63-linked chains serve nonproteolytic roles in intracellular protein trafficking (Clague and Urbe, 2006), transcription (Weake and Workman, 2008), DNA repair (Huang and D'Andrea, 2006) and kinase signaling (Chen, 2005). By contrast, little is known about the functionality of the non-canonical ubiquitin linkages (ubiquitin chains linked via lysines other than K48 and K63). The goal of this study is to elucidate the function of Lys29-linked ubiquitin chains by characterizing DUBs that disassemble Lys29-linked ubiquitin chains and by identifying substrates that undergo this modification.